Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in Kinshasa Province, Democratic Republic of Congo

Reports suggest non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa but their epidemiology is ill-defined, particularly in highly malaria-endemic regions. We estimated incidence and prevalence of PCR-confirmed non-falciparum and Plasmodium falciparum malaria infections within a longitudinal study conducted in Kinshasa, Democratic Republic of Congo (DRC) between 2015-2017. Children and adults were sampled at biannual household surveys and routine clinic visits. Among 9,089 samples from 1,565 participants, incidences of P. malariae, P. ovale spp., and P. falciparum infections by 1-year were 7.8% (95% CI: 6.4%-9.1%), 4.8% (95% CI: 3.7%-5.9%) and 57.5% (95% CI: 54.4%-60.5%), respectively. Non-falciparum prevalences were higher in school-age children, rural and peri-urban sites, and P. falciparum co-infections. P. falciparum remains the primary driver of malaria in the DRC, though non-falciparum species also pose an infection risk. As P. falciparum interventions gain traction in high-burden settings, continued surveillance and improved understanding of non-falciparum infections are warranted.

Also please clarify how the differences in prevalences were calculated.Page 9 of the methods indicates that binomial GEE was used but was one adjusted model used?Or was each risk factor (presented in Figure 5) evaluated on its own?Please add this detail.Discussion Pages 17-18: "The high proportion of P. malariae-infected participants who experienced multiple infections in the study (24%) is notable given that this species is known to recrudesce."What evidence is there for recrudescence of P. malariae infections?Vivax and ovale infections are known to relapse as they have dormant liver stages, but malariae infections do not have hypnozoites.Please elaborate on this statement and provide references for the assertions made.Table 2: The title of the table is slightly misleading; it only mentions non-falciparum infection but falciparum infection is also presented.Can you amend to include mention of falciparum infection in the title please?Table 2: How is the baseline (and year 1 and year 2) at-risk population defined for the clinic subpopulation?Are these participants who were negative for the malaria species at the baseline household visit AND who had at least one clinic visit during the follow-up period?Or is this otherwise defined?Please add more details to footnote 2 for the clinic sub-population.Table 2 and Figure 3: I am having a hard time understanding how the number of new infections and the at-risk population compares between Table 2 and Figure 3.The numbers of infections do not seem to quite add up to the household plus clinic population.There are no household events reported during year 2 and the number of new infections during year 2 in panel b of Figure 2 does not equal those reported in Table 2. Please clarify.Supplemental Table 3: Are the characteristics included in this table those of the participants at the baseline household visit?And the PCR positive are those with any positive PCR at the clinic at any time during the follow up period?Some clarifying notes would be helpful.

Nature Communications -Response to Reviewers
Manuscript #: NCOMMS-23-14691A Title: Epidemiology of Plasmodium malariae and Plasmodium ovale spp. in Kinshasa Province, Democratic Republic of Congo

Responses to Reviewer Comments:
Note -page numbers for revisions refer to the revised, tracked-changes manuscript draft.Bold, underlined text indicates new additions.
In addition to changes made in response to reviewer feedback, we have incorporated several additional wording edits throughout the revised manuscript for improved clarity, alignment with required formatting specifications, grammatical changes, and updates as needed.These are not explicitly noted below for simplicity.
Reviewer #1 (Remarks to the Author): This is a thoroughly conducted and well-reported study of non-falciparum malaria in an important high-burden sub-Saharan African setting.The methods and results are shared clearly and the extension of the analysis beyond reports of incidence/prevalence into the varying presence of mixed and mono infections in different populations and the differences in parasite densities make this a useful contribution to the field.
Major comments 1. Reporting non-falciparum epidemiology without any mention of vivax malaria (beyond citations) was surprising.It would be useful to know why vivax malaria was not included in this study.

Author Reply:
Thank you for this comment.We did not test for Plasmodium vivax in this study since another study previously published by our group found a low prevalence of P. vivax in the same region of Kinshasa, DRC, using Demographic and Health Survey data from a similar time period, 2013-14 (Brazeau N et al., Nature Communications, 2021.doi:10.1038/s41467-021-24216-3). Risk factors of P. vivax infection were also assessed in this prior study.
We agree that we should better acknowledge these previously reported P. vivax prevalence estimates in our manuscript for context and comprehensiveness.We have updated our manuscript accordingly.
Revisions: (Pg.8)."P.vivax was not tested in this study as previously published estimates of P. vivax prevalence in Kinshasa Province from a similar time period (2013-14) indicated low prevalence in the region (0 to <2%) 39 ." Minor comments: 1. Line 99 -P.ovale acute symptoms given ability to relapse?
Author Reply: We have edited the manuscript to also include the clinical impacts attributed to P. ovale spp.infections.
Revisions: (Pg.5), "P.ovale spp.infections commonly result in low morbidity, although severe complications have been documented in case reports 28,29 .The clinical relevance of P. falciparum co-infection with non-falciparum species is also unclear…" 2. Line 134 -was net use as well as ownership assessed?
Author Reply: Bed net use (defined by participant self-report that they slept under a bed net the prior night) was assessed in risk factor models and reported in study results.We also measured and reported household bed net ownership, finding that 72.5% of participants at the baseline survey lived in a household that owned at least one bed net, while only 45.0% of the population reportedly slept under a bed net the prior night.Both metrics, bed net use and ownership, are reported for the survey population in Table 1.
We added minor edits to the manuscript to better highlight net ownership and use in the text.
Revisions: (Pg.7), "At baseline and follow-up surveys, household-level and individuallevel questionnaires were collected to ascertain demographic information, household characteristics (e.g., housing materials, possessions for wealth indicators, bed net ownership and use), health status and clinical data…" 3. Starting line 138 -clarify if DBS was taken for all or just those RDT pos?
Author Reply: Dried blood spots were collected for all participants, regardless of RDT result, at each baseline and follow-up survey, and also at each unscheduled symptomatic health clinic visit.We edited the manuscript to clarify this point.

Revisions:
(Pg. 7), " All participants were screened for malaria infection at each visit using a combination RDT...." (Pg.7), "Where consented, Dried blood spot (DBS) samples were also collected from all consenting participants at each visit using Whatman 3MM filter paper (Fisher Scientific, Fair Lawn, NJ USA), and were stored with desiccant at -20°C for future molecular testing…" 4. Around line 188 -clarify if/that the clinical catchment is the same as the sampled communities and how that is known Author Reply: Thank you for this comment.All households were located within the clinical catchment areas of the study clinics.For two of the included health areas, Bu (rural), and Kimpoko (peri-urban) the study clinics were the main clinics in each health area.Each of the villages in those two health areas were selected for the study because of their proximity to the study clinics to ensure they were included in the clinic catchment or within 5km distance away.For the urban health area, Voix de Peuple, more health clinics were available to participants, so one neighborhood was selected in the catchment of a study clinic.This clinic served a socioeconomically diverse segment of the neighborhood's population.
A total of 3 health clinics were included in the study.
We edited the manuscript to clarify the sampling and study clinics included.
Revisions: (Pg.6), "Three local health facilities were included in our study, one per health area.The chosen facility was the main government clinic in two of these health areas (Bu -rural, Kimpoko -peri-urban).In urban Voix de Peuple, one private clinic was selected due to the diverse patient population it served.Only households within the clinic catchment areas that were located within 5km from the clinic were eligible for the study." 5. Line 401 -citation for malariae known recrudescence?More so that ovale relapse?Author Reply: Thank you for this comment.This was an error on our part.We have edited our manuscript to characterize the ability of Plasmodium malariae to persist longterm at low parasite densities in the blood.Citations have also been added.

Revisions:
(Pg. 20), "The high proportion of participants with P. malariae infected participants who experienced had multiple infections infection detected more than once during the study (24%) is notable given that this parasite species can persist in the blood at low parasite density for long periods of time 25,42,43 .Chronic P. malariae infection may be associated with deleterious clinical outcomes…" (Pg 20), "Though we could not distinguish whether multiple P. malariae infections detected in an individual were due to chronic occurrences or acute re-infection events…" 6. Line 443 -and clinics were easily physically accessible to community members?
Author Reply: All study health clinics were near main roads and walkable from enrolled households.Villages included as sites in the study were selected so that households would be no more than 5km from the clinic.However, differences in household wealth, ownership or access to transportation, work status and care-taking responsibilities could impact differences in accessibility to the clinic for reasons other than distance.
We have edited the manuscript slightly to note this.
Revisions: (Pg.6), "Only households within the clinic catchment areas that were located within 5km from the clinic were eligible for the study."

Line 445 -what is very low density?
Author Reply: Thank you for this comment.We agree this is a subjective term and we have edited the manuscript to more concretely characterize low and very low parasite densities.
Revisions: (Pg.22), "Third, our duplex PCR assay detected low density infections, with a limit of detection between 1-10 p/µL, but would likely miss infections of parasite densities below this range, which are known to occur with P. malariae and P. ovale species.
Reviewer #2 (Remarks to the Author): Nature Communications Epi of Pm and Po in DRC review This is a well written article summarizing a basic yet useful epidemiological study of the prevalence and incidence of non-falciparum and falciparum infections over three years in the DRC.A few critical methodological details should be clarified, including the justification for calculation of period prevalence and the overall sampling.

Methods
1.It would be helpful to provide a bit more detail on the sampling, even though the authors note these are described in further detail elsewhere.For example, how were the villages selected?Was probability sampling used allowing for population-based estimates?
Author Reply: Thank you for this comment.See response to Reviewer 1 regarding selection of the urban neighborhood.We have edited the manuscript to provide more detail on sampling as requested.
All households are located within the clinical catchment areas of the study clinics.For two of the Bu (rural) and Kimpoko (peri-urban) health areas, the study clinics selected for the study are the main government clinics serving each health area.The included villages within those two health areas were selected for the study because of their proximity to the study clinics.These villages were either within the same village as the study clinic or within 5km distance away.For the urban Voix du Peuple health area, more health clinics were available due to the densely populated urban setting.We therefore selected the neighborhood of the catchment of a private study clinic that was used by the most diverse population in that neighborhood in terms of wealth.
Revisions: (Pg.6), "Three local health facilities were included in our study, one per health area.The chosen facility was the main government clinic in two of these health areas (Bu -rural, Kimpoko -peri-urban).In urban Voix de Peuple, one private clinic was selected due to the diverse patient population it served.Only households within the clinic catchment areas that were located within 5km from the clinic were eligible for the study." 2. Additionally, were visits at health facilities only from enrolled participants at the baseline household visit included?It appears this is the case but it would be very helpful to clarify these details more explicitly.
Author Reply: This is correct, we only collected samples and data during health facility visits by enrolled participants.In this study, included participants were those who had DBS samples from the baseline household enrollment visit.Clinic visits among participants who did not have a prior baseline visit and DBS sample were not included.We have clarified this in the manuscript.
Revisions: (Pg.8), "1) The "survey-based" analysis consists of participant survey data and clinical samples collected at active surveillance household visits for all those enrolled at baseline, and 2) the "clinic-based" analysis consists of participant clinical survey data and clinical samples collected from the subset of the survey-baseded population who had samples collected at the baseline household enrollment visit and who visited study clinics as-needed when symptomatic during follow-up (passive surveillance).
3. Page 9: "Prevalences of P. malariae, P. ovale spp., and P. falciparum were estimated overall, and by infection type, calculated as the number of infections detected out of the total number of visits in the study".Do you mean to say that "Prevalence of Plasmodium infection was estimated overall, and by species type (P.malariae, P. ovale spp., and P. falciparum)…."?
Author Reply: This is correct.We have revised the text to read "by species." Revisions: (Pg.10), "Prevalences of P. malariae, P. ovale spp., and P. falciparum were estimated overall, and by species infection across follow-up." 4. Also, this is a rather unusual definition of period prevalence.Period prevalence is typically defined as the number of people with the disease/condition at any time during the period.To me it does not make sense to use the number of visits as the denominator-why not simply the number of people included during the time period and the numerator is any infection during that time period?Please justify the unusual methodology used to calculate period prevalence.
Author Reply: This was initially calculated at the per-visit level to account for repeated sampling across time and the possibility of multiple infection events throughout follow-up.This method assessed the number of infection events, rather than the number of subjects infected regardless of multiple infections experienced.In doing so, this method therefore assumed all infections were acute events, rather than the same ongoing infections detected at multiple screening points.We have removed the terminology of 'period prevalence' to avoid confusion in the denominator used.We have also updated the manuscript to report prevalence as suggested following typical convention, as the number of participants ever infected out of the total number of participants in each population.We also kept characterization of the proportion of all tested samples that were positive for an infection across all follow-ups, to distinguish infected participants vs. total number of infections detected during the approximately 3-year follow-up period.
Revisions: (Pg.10), "Prevalences of P. malariae, P. ovale spp., and P. falciparum were estimated overall, and by species infection across follow-up.calculatedas the total number of infections detected out of the total number of visits in the study.Factors associated with non-falciparum infection prevalences were evaluated using crude linear binomial generalized estimating equation (GEE) models accounting for repeated testing of participants over time." (Pg. 14-15), "Crude prevalence of non-falciparum infection was lower in household survey visits than symptomatic clinic visits, as expected.5. Also please clarify how the differences in prevalences were calculated.Page 9 of the methods indicates that binomial GEE was used but was one adjusted model used?Or was each risk factor (presented in Figure 5) evaluated on its own?Please add this detail.
Author Reply: Prevalence differences were calculated from linear binomial GEE models, accounting for repeated visits over time by participants.Prevalence difference models were all run individually as unadjusted models only, to report the crude association between each individual factor and the outcome of P. malariae, P. ovale, or P. falciparum infection.We have clarified in the manuscript that these were crude associations only, modeled individually.We also note in the discussion that there are no assumptions of causality in this study, therefore risk factor results should be interpreted only as associations.
Revisions: (Pg.10), "Factors associated with non-falciparum infection prevalences were evaluated using crude linear binomial generalized estimating equation (GEE) models accounting for repeated testing of participants over time.Possible factors associated with malaria infection were modeled individually.An exchangeable working correlation matrix was assumed for GEE models, and robust standard errors were estimated for calculation of 95% confidence intervals (CIs).
In clarifying the methods for modeling these associations, we also added discussion of the rationale for use of GEE models in this analysis rather than a generalized linear mixed model that would account for subject-level clustering within households, and householdlevel clustering within the 7 villages of the study design.Modeling this nested clustering structure is too complex given the low number of outcome events that were detected in the study for P. malariae and P. ovale spp., therefore GEE models were used that addressed the repeated nature of testing across follow-up.
Revisions: (Pg.10), "While three-level clustering of participants nested within households, and households within village sites is present in the study 31 , this clustering structure was too complex to account for using mixed-effect modeling because of the low number of P. malariae and P. ovale outcomes detected in this study.Therefore, the GEE model was used.As a sensitivity analysis, we compared GEE results for P. falciparum outcomes (which occurred frequently in the study) to results from a generalized linear mixed model accounting for random effects of clustering and repeated testing of subjects over time (Supplementary Table 3).

Discussion
Pages 17-18: "The high proportion of P. malariae-infected participants who experienced multiple infections in the study (24%) is notable given that this species is known to recrudesce."What evidence is there for recrudescence of P. malariae infections?Vivax and ovale infections are known to relapse as they have dormant liver stages, but malariae infections do not have hypnozoites.Please elaborate on this statement and provide references for the assertions made.
Author Reply: Thank you for this comment.See response to Reviewer 1 --this was an error on our part.We have edited the manuscript to properly characterize the ability of Plasmodium malariae to persist long-term at low parasite densities in the blood.Citations have also been added.

Revisions:
(Pg. 20), "The high proportion of participants with P. malariae infected participants who experienced had multiple infections infection detected more than once during the study (24%) is notable given that this parasite species can persist in the blood at low parasite density for long periods of time 25,42,43 .Chronic P. malariae infection may be associated with deleterious clinical outcomes…" (Pg 20), "Though we could not distinguish whether multiple P. malariae infections detected in an individual were due to chronic occurrences or acute re-infection events…"

Table 2 :
The title of the table is slightly misleading; it only mentions non-falciparum infection but falciparum infection is also presented.Can you amend to include mention of falciparum infection in the title please?